Adjuvants are materials that can be used either for the developments of vaccines having increased antigenicity or for therapeutic and preventive purposes by enhancing non-specific immune responses to antigens. Because adjuvants function to maintain strong and rapid immune responses to antigens for a long time when the antigenic levels are low, these adjuvants are used in the preparation of vaccines. Also, the adjuvants allow special antigens to be used or the levels of antigens to be changed, thereby regulating immune responses to the antigens or controlling the types and subclasses of antibodies against the antigens. In addition, the adjuvants can be used to enhance immune responses, particularly in immunologically immature or senescent persons, in order to enhance the induction of mucous immunity.
Most of adjuvants were found in many natural materials through many trials and errors. In the first worldwide report on the adjuvants, in 1925, Ramon (France) reported that tapioca starch (Casaba) which is used in foods was mixed with diphtheria and tetanus toxoid, antigenic specificity and antibody production were effectively increased. Since then, the immune-enhancing effect of an aluminum adjuvant was reported, and an effective emulsion-type adjuvant containing inactivated killed mycobacteria as an immune modulator was developed. It is Freund's complete adjuvant (FCA) known as a very effective immune modulator, but was not suitable for human use because of its high reactogenicity. For this reason, Freund's incomplete adjuvant (FIA) containing no mycobacteria was developed and approved in Britain. Gram-negative bacterial endotoxin was reported to have an immune-enhancing effect, and the effect of muramyl dipeptide (MDP) was confirmed by Ellouz et al. in 1974 (Ellouz F. et al., Biochem. Biophys. Res. Coomun. 59:1317-25, 1974). Since then, it was reported that lecithin, saponin and the like can also be used as adjuvants for enhancing immunity.
An ideal adjuvant should have an immune-enhancing effect and should also be nontoxic, highly biodegradable, easy to use, easy to be available and inexpensive. Until now, many types of adjuvants have been reported, but only several types of adjuvants can be actually used in clinical practice. This is because reliable research data on safety that is the most important in the development of adjuvants for use in vaccines should be supported.
Vaccines have therapeutic and preventive effects, and thus can reduce the incidence rate of disease to 99%. Thus, vaccines are medicines that have a high effect versus cost. These days, the use of vaccines is not limited only to infectious diseases, but is being widened to various intractable diseases, including cancer and autoimmune diseases. Also, as therapeutic vaccines emerge, the development of vaccines is being recognized to be very important. Thus, the development of adjuvants as vaccine-related products is being accelerated together with the development of vaccines. As the range of immune-related diseases widens, the development of new adjuvants is being recognized as a very promising field.
Meanwhile, the present inventors acquired a patent relating to a high-molecular-weight poly-gamma-glutamic acid and the use thereof (Korean Patent Registration No. 399091), and a patent relating to a method of producing poly-gamma-glutamic acid using Bacillus subtilis var. chungkookjang, a salt-tolerant strain producing high-molecular-weight poly-gamma-glutamic acid (Korean Patent Registration No. 500796), as well as patents relating to an anticancer composition, an adjuvant, an immune-enhancing agent, and inhibition of viral infection (Korean Patent Registration Nos. 496606, 517114, 475406 and 0873179). In addition, the present inventors reported a hyaluronidase inhibitor containing poly-gamma-glutamic acid (Korean Patent Registration No. 582120) and found the anticancer effect based on immune-enhancing effect of poly-gamma-glutamic acid [Poo, H. R. et al., Journal of Immunology, 178:775, 2007, Poo, H. R. et al., Cancer Immunol Immunother (published online: 18 Mar. 2009)]. That is, the present inventors performed extensive studies to develop the use of poly-gamma-glutamic acid, including the medicinal use of poly-gamma-glutamic acid, thereby finding the various effects of poly-gamma-glutamic acid.
Meanwhile, polymeric nanoparticles, particularly nanoparticles made of biodegradable polymers such as poly-caprolactone, are receiving a great deal of attention due to their high biocompatibility. However, these nanoparticles have a shortcoming in that they are not suitable for delivery of hydrophilic drugs or antigens, because they are hydrophobic in nature.
Examples of the use of poly-gamma-glutamic acid for the in vivo delivery of proteins or the enhancement of humoral immunity were reported (Akagi, T. et al., J. controlled release, 108:226, 2005; Uto, T. et al., the J, Imunol., 178:2979, 2007). However, when poly-gamma-glutamic acid alone is used as an adjuvant, it has insufficient ability to produce an antibody. Thus, the ability of poly-gamma-glutamic acid to induce antigen-specific immunity needs to be further enhanced.
Chitosan is a cationic polysaccharide that is a deacetylated form of chitin and it is nontoxic and highly biocompatible. Also, chitosan is known as a material that can open the tight junction between cells, and thus is highly effective in mucosal drug delivery systems. Most chitosans have a molecular weight of 50-2,000 kDa and dissolve in an acetic acid solution (pH 4). However, in order to allow chitosan to be applied as medicinal materials, chitosan should be kept in an aqueous solution at neutral pH. In order to maintain chitosan in a cationic aqueous solution at physiological pH, treating chitosan with cellulase to reduce the molecular weight is necessary.
A complex of poly-gamma-glutamic acid and chitosan nanoparticles is an ionically bonded complex of poly-gamma-glutamic acid and chitosan and is used either as a carrier for oral delivery of insulin or DNA delivery, but the use thereof in the induction of immune responses was not reported (Lin, Y. et al., Biomacromolecules, 6:1104, 2005; Lin, Y. et al., Nanotechnology, 16:105102, 2007).
Accordingly, the present inventors have made extensive efforts to overcome the above-described problems occurring in the prior art and, as a result, have found that, when an adjuvant containing nanoparticles prepared by ionic bonding between poly-gamma glutamic acid and chitosan is administered to mice together with various antigens, the production of antibodies significantly increases compared to when poly-gamma-glutamic acid alone is used as an adjuvant, thereby completing the present invention.